In 32PO4-labeled rat adipocytes, isoproterenol (ISO), adenosine deaminase (ADA), or insulin rapidly increased phosphorylation of a particulate 135 kDa protein which was identified as native cGMP-inhibited "low Km" cAMP phosphodiesterase (cGI PDE) and was selectively immunoprecipitated from solubilized particulate fractions with anti-cGI PDE IgG. The time course and concentration dependencies for phosphorylation and activation of cGI PDE were similar for ADA, ISO and insulin; maximal agonist-dependent labeling was similar for all three agents. A particulate 135 kDa phosphoprotein was also isolated from 32PO4-labeled 3T3-L1 differentiated adipocytes incubated with ISO or insulin. In fat adipocytes incubated with ADA, phenylisopropyladenosine (PIA) (an ADA-resistant adenosine analog) prevented or reversed both phosphorylation and activation of cGI PDE, and inhibited the stimulatory effects of ADA on cAMP-dependent protein kinase (A-kinase) and lipolysis. Incubation of rat adipocytes with insulin in the presence of ADA or ISO transiently evoked about 40-300% greater phosphorylation and activation of cGI PDE than the added effects of insulin and lipolytic agent alone; activation of cGI PDE preceded insulin-dependent decreases in A-kinase and lipolysis. These and other results suggest that ADA and beta-agonist phosphorylate and activate cGI PDE via A-kinase; that the antilipolytic effect of insulin is associated with phosphorylation/activation of cGI PDE via an unidentified intracellular serine kinase, and does not require adenosine-mediated inhibition of adenylate cyclase; and that regulation by Gi (the guanine nucleotide binding protein which inhibits adenylate cyclase) is important for the cAMP-dependent activation of hormone-sensitive PDE.